Purification and isotype analysis of protein kinase C from rat liver nuclei

The International Journal of

PERGAMON

The International Journal of Biochemistry & Cell Biology 30 (1998) 185±195

Biochemistry & Cell Biology

Puri®cation and isotype analysis of protein kinase C from rat liver nuclei MarieÈlle P. de Moel, Sjenet E. Van Emst-De Vries, Peter H.G.M. Willems, Jan Joep H.H.M. De Pont * Department of Biochemistry, Institute of Cellular Signalling, University of Nijmegen, P.O. Box 9101, 6500 HB Nijmegen, The Netherlands Received 3 March 1997; accepted 19 September 1997

Abstract The properties and subtype composition of protein kinase C present in rat liver nuclei were studied in a Triton-X100 extract of isolated puri®ed nuclei. The enzyme activity was dependent on both Ca2+ and phosphatidylserine, but the phorbol ester 12-O-tetradecanoylphorbol 13-acetate gave only a partial stimulation. Both histone and myelin basic protein served as substrate. Puri®cation of the Triton-X-100 extract followed by Q-Sepharose chromatography gave a preparation with a speci®c activity of 70 pmol/mg protein min. Western blotting of this preparation showed only the presence of the d and z subtypes, but not the a-subtype, although the latter was present in rat liver homogenates. The b, g and E subtypes were not found in the homogenate nor in the nuclear extract. The speci®c activity of protein kinase C could be further increased up to 800 pmol/mg protein min after protamine agarose chromatography. Also in this preparation the presence of the d and z subtypes could be established. # 1998 Elsevier Science Ltd. All rights reserved. Keywords: Nucleus; Protein kinase C

1. Introduction Protein kinase C (PKC), a family of phospholipid-dependent serine/threonine kinases, plays an Abbreviations:ATP,adenosinetriphosphate,DAG,diacylglycerol, DiSBAC2(3), bis-(1,3-diethylthiobarbituric acid)trimethine oxonol, DTT, 1,4-dithiothreitol, EGTA, ethylene glycol bis(b aminoethyl ester) N,N'-tetraacetic acid, PAGE, polyacrylamide gel electrophoresis, PBS, phosphate bu€ered saline, PKC, protein kinase C, PMSF, phenylmethylsulfonyl ¯uoride, PtdSer, phosphatidylserine, TPA, 12-O-tetradecanoylphorbol 13-acetate. * Corresponding author. Tel.: +31-24-3614260; fax: +3124-3540525; e-mail: [email protected]

important role in the signal transduction pathway at the plasma membrane level and mediates a wide range of cellular responses [1]. Since the discovery of the protein a large series of new members of the family have been found in which one can distinguish three subgroups based on the domain structure and the biological activity [2, 3]. All members have a kinase domain in common but are di€erently regulated. Conventional PKC subtypes a, b and g (CPKC) are Ca2+ and phospholipid dependent and either diacylglycerol or phorbol esters can stimulate their enzymatic activity. PKC subtypes d, E, Z (L) and y (novel PKCs, nPKCs) are not Ca2+ dependent but need

1357-2725/98/$19.00 # 1998 Elsevier Science Ltd. All rights reserved. PII: S 1 3 5 7 - 2 7 2 5 ( 9 7 ) 0 0 1 2 2 - 2

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both phospholipid and phorbol ester for enzymatic activity. The atypical PKC isozymes PKCz and PKC-l (aPKCs), are only phospholipid dependent and do not need the presence of Ca2+ and phorbol ester. For the di€erent isotypes speci®c, tissue and cellular, expression [4], activation requirements and preferential substrates have been demonstrated [5±7]. In rat liver nuclei the presence of PKC-b was reported by Rogue et al. [8]. In contrast Alessenko et al. [9] found that the isotypes a and d were present both in the cytosol and in the nucleus. The latter ®nding was in agreement with the report of Wetsel et al. [4] who reported the presence of these isotypes in rat liver homogenate. In addition they reported the presence of PKC-z in this tissue, but these authors did not study the nuclear localization. Recent studies indicate that many elements involved in the receptor mediated signal transduction pathway are also present in either the nucleus or in the nuclear membranes. For example, Ca2+ concentrations in the nucleus and the cytosol di€er from each other and changes in these concentrations do not correlate necessarily [10±14]. In the nuclear envelope a Ca2+-ATPase similar to that in the endoplasmatic reticulum has been demonstrated but the mechanism involved in Ca2+ bu€ering in the nucleus is not yet clear [15, 16]. Elements of the phosphoinositide cycle have also been demonstrated in nuclei after stimulation of whole cells [17, 18] or puri®ed nuclei [19±22]. The di€erential localisation of phosphoinositide kinases suggests that phosphoinositide metabolism may play a speci®c role in the nucleus [23, 24]. Enzymes like phospholipase Cb [25, 26] and phospholipase A2 are also found in nuclei. A nuclear diacylglycerol pool di€erently regulated compared to the plasma membrane pool has also been demonstrated by various groups [21, 27, 28]. Originally, the phosphorylation of proteins in the nucleus was thought to be caused by translocation of PKC from the cytosol into the nucleus [29]. Recently, however, several groups have presented evidence for the nuclear localization of PKC in di€erent tissues under basal

conditions [30±32] or after stimulation of cells [33±36]. Until now knowledge on PKC types present in the nucleus of various cell types is limited and controversial. The kinetic properties of the nuclear enzyme have not been studied in detail. We therefore isolated rat liver nuclei and extracted the protein kinases in order to determine its kinetic properties and to analyze which subtypes are present. We could indeed demonstrate PKC activity in the nuclear extract of rat liver and established the presence of the subtypes protein kinase Cd and z in rat liver nuclei. 2. Materials and methods 2.1. Materials EGTA was purchased from Merck (Darmstadt), PMSF from Serva (Heidelberg), Triton-X-100 from BDH (Poole), I-block from Tropix (Bedford, MA), Q-Sepharose (pre-packed column) from Pharmacia (Uppsala) and PtdSer from Lipid products (Redhill). DTT, NBT and BCIP were obtained from Research Organics (Cleveland, OH). Membrane ®lters (ME28 1.2 mm) were purchased from Schleicher and Schull (Dassel), PVDF blotting membrane from Millipore (Bedford, MA), ATP from Boehringer (Mannheim) and [g-32 P]-ATP from Amersham (Buckinghamshire). Trypsin inhibitor, leupeptin, histone IIIS, TPA, protamine agarose, alkaline phosphatase labeled goat anti-rabbit IgG and polyoxyethylene sorbitan monolaurate (Tween20) were all obtained from Sigma (St. Louis, MO). Isozyme speci®c antibodies to PKC were purchased from Life Technologies (Gathersburg, MD). These monospeci®c polyclonal antibodies were raised in rabbit against the following sequences: AGNKVISPSEDRRQ (AA 313±326) for PKC-a, GPKTPEEKTANTISKFD (AA 313±329) for PKC-b, NYPLELYERVRTG (AA 306±318) for PKC-g, SFVNPKYEQFLE (AA 662±673) for PKC-d, KGFSYFGEDLMP (AA 726±737) for PKC-E and GFEYINPLLLSAEESV (AA 577±592) for PKC-z, respectively.

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The activity of these antibodies was con®rmed by a positive reaction in rat brain homogenates, known to contain all these six PKC isotypes, with the appropriate antibody.

2.2. Isolation of nuclei and control of purity Nuclei were isolated from rat liver essentially as described by Blobel and Potter [37]. Fresh rat livers were used and all steps were carried out at 48C. Livers were cut into small pieces, washed twice to remove blood and homogenized in homogenisation bu€er (250 mM sucrose, 50 mM Tris±HCl pH 7.4, 0.2 mM EGTA, 10 mM MgCl2, 5 mM DTT, 1 mM PMSF, 0.2 mg/ml trypsin inhibitor and 20 mM leupeptin) in a Potter±Elvehjem homogenizer with loose ®tting pestle. After ®ltration through nylon gauze the homogenate was made up to 40 ml/liver. The pellet obtained after two sequential centrifugation steps at 1000g for 10 min was resuspended in a 2.3 M sucrose bu€er (2.3 M sucrose, 50 mM Tris±HCl pH 7.4, 0.2 mM EGTA, 10 mM MgCl2, 5 mM DTT, 1 mM PMSF) and loaded on 2.1 M sucrose bu€er with a GTEMS cushion (40% glycerol with 2.3 M sucrose buffer). The white nuclear pellet after ultracentrifugation at 73,000g for 1 h was resuspended in the GTEMS cushion, washed two times and diluted with homogenisation bu€er to a concentration of 5*107 nuclei/ml. This nuclear suspension was checked for plasma membrane and endoplasmatic reticulum contamination by determination of Na+,K+ATPase [38] and NADPH-cytochrome C reductase [39] activities, respectively. The quality of the nuclei was tested by confocal laser scan microscopy. This was performed by incubating the nuclear pellet with DiSBAC2(3) and transferring it onto coverslips. Preparations were examined in a Leica confocal laser scanning microscope equipped with a 63  /1.4 N.A. oil immersion lens. A 488-nm laser was used to excite DiSBAC2(3) and a 510±560 nm bandpass ®lter was placed in front of the detector. The average of four scans was taken and digitalized.

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2.3. Partial puri®cation of protein kinase C from nuclei In order to prepare a nuclear homogenate, the nuclear suspension was brought to a 0.1% Triton-X-100 concentration, sonicated at 100 W three times for 20 s and left on ice for 20 min. The supernatant after centrifugation at 100,000g for 20 min was de®ned as nuclear extract. Portions of 25 ml (4.4 mg protein) of this extract were applied on a Q-Sepharose column (5 ml, equilibrated with bu€er A (250 mM sucrose, 50 mM Tris±HCl, 0.2 mM EGTA, 5 mM DTT, 1 mM PMSF and 0.01% Triton-X-100)) connected to a FPLC system (Pharmacia, Uppsala). The protein was eluted with a linear gradient of 0±300 mM KCl in bu€er A, ¯ow 2 ml/min. PKC activity of the fractions was measured and the peak fractions were pooled. PKC was eluted from this column at KCl concentrations between 120 and 160 mM. All pooled fractions from Q-Sepharose were collected (6.0 mg protein) and adjusted to 0.6 M KCl in bu€er A and applied to a protamine±agarose column (1 ml, equilibrated with 0.6 M KCl in bu€er A; ¯ow 0.6 ml/min). Sequential elution was performed with 6 ml 0.6 M KCl in bu€er A; 2.4 ml 0.5 M KCl, 1 mM ATP and 10 mM MgCl2 in bu€er A; 2.4 ml 0.6 M KCl, 1 mM ATP and 10 mM MgCl2 in bu€er A; 2.4 ml 0.7 M KCl, 1 mM ATP and 10 mM MgCl2 in bu€er A; 2.4 ml 1.5 M KCl, 1 mM ATP and 10 mM MgCl2 in bu€er A. In these fractions PKC activity was also measured and the peak fractions were pooled. 2.4. Measurement of PKC activity In general 20 ml of a sample was incubated in a total volume of 200 ml containing 250 mM sucrose, 50 mM Tris±HCl pH 7.5, 10 mM MgCl2, 30 mM CaCl2, 50 mM [g-32 P]-ATP (2.5 mCi/ml), 50 mg/ml histone IIIS and 0.5 mM PMSF. Under activating conditions 100 mg/ml PtdSer (sonicated for 5 min) and 50 nM TPA were present. Phosphorylation was started by adding sample to the reaction mixture and incubations were performed at 308C for speci®ed

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times. Assays were stopped by addition of 5 ml 5% TCA with 100 mM H3PO4 and ®ltered over a membrane ®lter. Filters were washed and radioactivity was determined by Cerenkov radiation measurement in Opti-¯uor (Packard). PKC activity was de®ned as the amount of phosphorylation in medium with Ca2+, PtdSer and TPA minus the amount of phosphorylation in medium with Ca2+ alone. 2.5. Western immunoblotting Samples were separated on a SDS/10% polyacrylamide gel according to the method of Laemmli [40]. Gels were electroblotted to PVDF membranes and blocked for two h with block bu€er (0.2% I-block reagent and 0.1% Tween-20 in PBS). Slices were cut and incubated individually overnight each with an anti-PKC antibody (1:500 in wash:block 1:1) speci®c for one of six isotypes (PKC-a, PKC-b, PKC-g, PKC-d, PKC-E and PKC-z) as indicated. Parallel incubations with antibody and matching peptide (1:500 and 1:1000 in wash:block 1:1, respectively) were performed. After washing with washing bu€er (0.3% Tween-20 in PBS) the slices were incubated for 2 h with alkaline phosphatase conjugated goat anti-rabbit second antibody (1:1000 in wash:block 1:1). Blots were washed several times with washing bu€er and ®nally with PBS. The blots were colored with p-nitroblue tetrazolium chloride (NBT)/5-bromo-4-chloro-3-indolyl phosphate di(2-amino-2-methyl-1,3 dipropenediol) salt (BCIP). 2.6. Other procedures Protein concentrations were determined as described by Bradford [41] using bovine serum albumin as a standard. A cytosolic fraction of rat liver was prepared from the same homogenate as the nuclei. The supernatant after the ®rst centrifugation step was taken and centrifuged at 100,000g for 1 h. The supernatant was applied to a Q-Sepharose column and eluted according to the same conditions as described for the nuclear extract. PKC-activity

was measured and the peak fractions were pooled and used for Western blotting. 3. Results Nuclei were isolated from rat liver by puri®cation through a sucrose layer. In total 29 rat livers were used to purify nuclei. The purity of the nuclei was checked by determination of the activities of the plasma membrane marker, Na+,K+-ATPase, and the endoplasmatic reticulum marker, NADPH cytochrome C reductase, in the nuclear suspension. Less than 1% of either of the two activities in the homogenate was present in the nuclear suspension. In addition the nuclei were stained with the ¯uorescent membrane probe DiSBAC2(3) and analyzed by confocal laser scanning microscopy and were found to be pure (Fig. 1). From a nuclear suspension (5*107 nuclei/ml) a 0.1% Triton-X-100 extract was made. We ®rst studied the kinetic properties of the protein kinases present in the nuclear extract. The activity was measured in the presence of either Ca2+ or the combination of Ca2+, PtdSer and TPA. The di€erence between the measured activities under these two conditions was de®ned to be caused by the PKC activity present in the extract. Under standard conditions of 100 mg/ml PtdSer, 50 nM TPA, 50 mg/ml histone IIIs and 30 mM CaCl2, the PKC activity was linear with time during 20 min (not shown). We therefore used in further studies incubations of maximally 15 min. From the ATP-dependency curve (not shown) a Km value for ATP of 15 mM was calculated. In further experiments 50 mM ATP was used. The Ca2+ dependency of PKC activity showed a bell-shaped pattern with an optimum at 100 mM Ca2+ (Fig. 2(a)). The stimulatory PtdSer e€ect was maximal at 30 mg/ml, but was not decreased at higher concentrations (Fig. 2(b)). The stimulatory curve of the e€ect of TPA was rather ¯at (Fig. 2(c)). The comparison of phosphorylation experiments with either histone or myelin basic protein (MBP) as substrate (Fig. 3(a) and (b)), shows a higher maximal activity with MBP. The maximal

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Fig. 1. Confocal laser scan micrograph of isolated rat liver nuclei. Rat liver nuclei were isolated as described in Section 2 stained with DiSBAC2(3) and visualized on a Leica confocal laser scanning microscope. Images were averaged over four scans.

activity was reached at a lower concentration with the latter substrate (10 mg/ml with MBP and 50 mg/ml with histone). A di€erence in the molecular mass of the substrate proteins and the number of phosphorylation sites per molecule could at least in part explain the di€erence in maximal activities with the two substrates. With MBP as substrate no inhibition at higher substrate concentrations (up to 60 mg/ml) was found. With histone as substrate concentrations up to 300 mg/ml could be used and in this case a marked inhibition of PKC activity at substrate concentrations of 75 mg/ml and higher was observed. Histone seemed to be a good substrate for Ca2+-dependent phosphorylation, while with MBP the Ca2+-dependent phosphorylation parallels the Ca/PtdSer/TPA stimulated phosphorylation at MBP concentrations of 5 mg/ml or

higher (Fig. 3(b)). Comparison of two di€erent substrates, however, is rather complicated, since the number of phosphorylation sites on each protein is unknown. To determine which isotypes of PKC are present in the nuclear extract we ®rst analyzed which isotypes were present in a rat liver homogenate and cytosol. In a rat liver homogenate PKC-a, PKC-d and PKC-z were found (not shown). To determine which isotypes of PKC were present in the cytosol, a cytosolic fraction was puri®ed on a Q-Sepharose column and the fractions with PKC-activity were pooled. Also in the cytosol the same three isotypes were found to be present: PKC-a, PKC-d and PKC-z (Fig. 4). PKC-b, PKC-g and PKC-E could not be detected in the cytosol using speci®c antibodies for these subtypes. With all these antibodies positive

Fig. 2. Enzymatic properties of protein kinase C activity in a Triton-X-100 extract of isolated rat liver nuclei. The protein kinase activity was measured in 20 ml samples of the nuclear extract. In general the activity was measured in a medium as described in Section 2 with exception of the following. In (A) the Ca2+ concentration, in (B) the PtdSer concentration and in (C) the TPA concentration varied as indicated in the ®gure. The activity in the presence of Ca2+, PtdSer and TPA is given in open squares and the activity with Ca2+ alone in open triangles. The di€erence between these two activities, de®ned as protein kinase C activity, is given in open circles. The lines through the symbols are drawn by hand.

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Fig. 3. Substrate dependency of protein kinase C activity in a Triton-X-100 extract of isolated rat liver nuclei. The protein kinase activity was measured in 20 ml samples of the nuclear extract. In general the activity was measured in a medium as described in Section 2 with either histone (A) or myelin basic protein (MBP; B) in the indicated concentrations as substrate. The activity in the presence of Ca2+, PtdSer and TPA is given in open squares and the activity with Ca2+ alone in open triangles. The di€erence between these two activities is given in open circles. The lines through the symbols are drawn by hand.

Fig. 4. Western blot from Q-Sepharose puri®ed rat liver cytosol. Rat liver cytosol was puri®ed over a Q-Sepharose column as described in Section 2. 1 mg of protein from the pooled peak was applied to a large slot on a SDS-PAGE gel and blotted after running. Slices of the blot were incubated with PKC isotype-speci®c antibodies: in lane 1 with anti-PKCa, in lane 3 with anti-PKCb, in lane 5 with anti-PKCg, in lane 7 with anti-PKCd, in lane 9 with anti-PKCE and in lane 11 with anti-PKCz. In parallel the slices were incubated with the same antibodies and their matching peptides, lanes 2, 4, 6, 8, 10 and 12, respectively. On the left the positions of the molecular weight standards are indicated and on the right the expected position of PKC (280 kDa) is marked.

results were obtained with a rat brain homogenate (not shown), indicating that all antibodies were reactive in the system used. Of the nuclear extract obtained by Triton-X100 extraction 400 ml was used for sixteen sequential elutions on a Q-Sepharose column. The eluted fractions were pooled and the PKC activity was measured in these collected fractions. A typical example of the elution pro®le is shown in Fig. 5. In this experiment fractions (50±54) with the highest PKC-activity (270 pmol/mg/ min) were pooled. From this partially puri®ed nuclear extract a Western blot was prepared and the isotypes present were analyzed. As shown in Fig. 6, PKC-a could not be detected in QSepharose puri®ed nuclear extract, but both PKC-d and z were indeed present. In addition to the 80 kDa bands representing PKC-d and z, the blot shows a band at 58 kDa which reacts with the antibodies against both PKC-d and z, but which could not be displaced by the speci®c pep-

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indeed be established. Like in Fig. 6 the blot also showed an 58 kDa band reacting with both PKC-d and PKC-z which could not be displaced by speci®c peptides. The 55 kDa band reacting with PKCz, as seen in Fig. 6, was absent on this blot, suggesting that it was separated from PKC in the protamine puri®cation step.

4. Discussion

Fig. 5. Elution pro®le of a nuclear extract on a Q-Sepharose column. Elution was performed with 0±300 mM KCl in bu€er A (see Section 2) as indicated (thick line). PKC-activity (open squares) and protein concentration (open circles) were determined in 20 ml fractions as described in Section 2.

tides. In addition the antibody against PKCz gave an apparent speci®c reaction with a protein of 55 kDa, the origin of which is unknown. PKCb, PKCg and PKCE could neither be detected in this partially puri®ed extract (not shown). To further purify the nuclear extract 56 ml from the pooled Q-Sepharose fraction was brought with KCl to 0.6 M (total 68 ml) and applied to a protamine agarose column. The elution pro®le and the PKC-activities are shown in Fig. 7. Fractions 66±67 contained the highest PKC-activity (2800 pmol/mg/min) and were pooled. The speci®c PKC activity after this puri®cation step was about eleven times higher than after the Q-Sepharose puri®cation. To verify whether the same isotypes of PKC were present in this protamine agarose fraction a Western blot was again prepared. Slices of the blot were incubated with anti PKC-a, anti PKCd and anti PKC-z (Fig. 8) and the presence of both PKC-d and PKC-z in this fraction could

The present paper shows that it is possible to extract with Triton-X-100 protein kinase C in an active form from isolated rat liver nuclei. The protein kinase C activity was de®ned as the di€erence between the activities in the presence of the combination of Ca2+, PtdSer and the phorbol ester TPA minus the activity in the presence of Ca2+ alone. The activity needed the presence of 0.1 mM Ca2+ and 25 mg/ml PtdSer. Although the activity was slightly increased by the addition of TPA and the maximal activity was reached with 0.1 mM TPA there was considerable activity in the absence of the phorbol ester. Both histone and myelin basic protein were good substrates for this enzyme.

Fig. 6. Western blot of Q-Sepharose puri®ed nuclear extract. 1 mg protein of the pooled Q-Sepharose fractions with the highest protein kinase C activity (fractions 50±54; Fig. 4) was applied to a SDS-PAGE gel and blotted after running. Slices were incubated with antibodies against PKCa (lane 1), PKCd (lane 3) and PKCz (lane 5). For lanes 2, 4 and 6 the matching peptides were also included during the incubation with the antibodies. On the left the molecular weights of the markers are indicated and on the right the expected position of PKC (280 kDa) is given.

M.P. de Moel et al. / The International Journal of Biochemistry & Cell Biology 30 (1998) 185±195

Fig. 7. Elution pro®le of the pooled Q-Sepharose puri®ed nuclear extract on the protamine agarose column. The pooled Q-Sepharose fractions with the highest protein kinase C activity (fractions 50±54 of Fig. 4) were applied to a protamine agarose column. When 0.6 M KCl in bu€er A was used as elution bu€er only protein (circles) but no protein kinase C (squares) came o€ the column. Immediately after application of 0.5 M KCl, 1 mM ATP and 10 mM MgCl2 in bu€er A (arrow) protein kinase C activity came o€ the column. When the column was sequentially eluted with higher salt concentrations no additional protein kinase C activity could be detected in the eluate (not shown).

Due to the interference of the detergent in the immunochemical assay it was not possible to determine the subtypes of protein kinase C in the Triton-X-100 extract. After Q-Sepharose puri®cation as well as after further puri®cation with a protamine agarose column only the presence of the d and z subtype could be established. These subtypes are both also present in rat liver homogenate and in a cytosolic extract. The a-subtype, however, which is also present in the rat liver cytosol could not be detected by immunoblotting. This is unexpected since this subtype is in contrast to the d and z subtypes, Ca2+ dependent and in the rat liver extract the protein kinase C activity needs the presence of this ion for activity.

193

There are several possibilities to explain this discrepancy. It might be that the a-subtype is also present in the nuclear extract but has been lost during the puri®cation step. This is unlikely since in a similar procedure with the cytosol the a subtype could be recovered. It might also be that there is a Ca2+-dependent protein kinase C of a subtype against which no antibody is yet available. It might also be that either the d or the z needs Ca2+ after extraction by Triton-X-100. In previous studies the presence of the bII-subtype in rat liver nuclei has been reported [8]. Goss et al. [42] identi®ed this activity as a mitotic lamin kinase. In the present study no b isotype could be found, neither in the cytosol nor in the nuclear extract. The bII subtype was also not found in rat liver nuclei by Alessenko et al. [9]. Whether this discrepancy must be attributed to the speci®city of the antibodies used in the di€erent studies is unclear. The present study indicates that at least the d and the z-subtype are present in rat liver nuclei. The d subtype was also found by Alessenko et al. [9]. Since the z-subtype has no binding site for phorbol esters it suggests that the limited sensitivity for phorbol esters of the activity in the

Fig. 8. Western blot of the protamine agarose puri®ed nuclear extract. 1 mg of the fractions of the protamine agarose column with the highest protein kinase activity (fractions 66 and 67 of Fig. 6) was applied to a SDS-PAGE gel and after running blotted. Slices of the blot were incubated with antibodies to PKCa (lane 1), PKCd (lane 3) and PKCz (lane 5). For lane 2, 4 and 6 the matching peptides were also included during the incubation with antibody. On the left the molecular weights of the markers are indicated and on the right the position of PKC (280 kDa) is given.

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extract is due to the large contribution of the zsubtype. The role of the various subtypes of protein kinase C in nuclear function is still unclear. Further investigations are necessary to give each subtype present a speci®c role.

Acknowledgements The study was supported by a grant of the Netherlands Organization for Scienti®c Research (NWO) through the Foundation for Life Sciences (SLW). The research of Dr. P.H.G.M.W. has been made possible by a fellowship of the Royal Netherlands Academy of Arts and Sciences. Dr. W. Schul and Dr. R. van Driel are gratefully acknowledged for their help with the isolation of the nuclei and the confocal microscopy.

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